Agent for preventing and curing hindrance of ischemic reperfusion

ABSTRACT

An agent for preventing and curing the hindrance of ischemic reperfusiton is disclosed which has as an active component thereof a chromanol glycoside represented by the following general formula:  
                 
 
     [wherein R 1 , R 2 , R 3 , and R 4  independently denote a hydrogen atom or a lower alkyl group, R 5  denotes a hydrogen atom, a lower alkyl group, or a lower acyl group, X denotes a monosaccharide residue or an oligosaccharide residue which may have a lower alkyl group or a lower acyl group substituted for the hydrogen atom of the hydroxyl group of the saccharide residue, n denotes an integer of 0-6, and m denotes an integer of 1-6]. The agent, even in a small dosage, acts safely and effectively on the affected part and allows the hindrances of ischemic reperfusion induced in heart, stomach, small intestine, liver, spleen, kidney, brain, and skin, and the hindrance induced during the transplantation of an internal organ to be prevented and cured.

TECHNICAL FIELD

[0001] This invention relates to a novel agent for preventing and curingthe hindrance of ischemic reperfusion. More particularly, the inventionrelates to an agent having a water-soluble chromanol glycoside as anactive component and used for preventing and curing the hindrance ofischemic reperfusion.

BACKGROUND ART

[0002] If the human cells and tissues are exposed to ischemic conditionfor long time, they suffer serious damage and eventually result in celldeath. On the other hand, when a sudden oxygen load is exerted thereonby an ischemic reperfusion for a certain duration, they sustain furtherserious hindrance. Clinically, these hindrance of the ischemicreperfusion have been heretofore recognized as diseases to be observedduring the transplantation of an internal organ or the reconstruction ofthe coronary blood flow for a myocardial infarct. In recent years, sincethe involvement of a free radical reaction in the histological hindrancedue to the ischemic reperfusion in a feline intestine was pointed out(Granger, D. N. et al.: Superoxide radicals in feline intestinalischemia. Gastroenterol. 22-29, 1981), the studies in this field havebeen widely disseminated and the involvement of active oxygen freeradical in the hindrance of ischemic reperfusion in not only the smallintestine but also the brain, heart, stomach, liver, kidney, and so onhas been appearing in reports, Therefore, numerous studies have beenbeing promoted with a view to seeking out ways of alleviating therelevant diseases by the administration of an exogenous radicaleliminating agent. However, owing to various problems concerning the invivo action and the field of reaction, however, all the radicaleliminating agents are not capable of effectively restraining thehistological hindrance due to the ischemic reperfusion (HirofumiKazumori et al.: Proposal of problems concerning the hindrance ofreperfusion and free radicals, J. Act. Oxyg. FreeRad.: 757-766, 1991).This fact is self-evident from a present state that virtually no radicaleliminating agent has been authorized as a drug.

[0003] The chromanol glycoside to be used in this invention is a knowncompound (the official gazette of JP-A-07-118,287). The chromanolglycoside is obtained by substituting an alcohol for the phytyl group atthe 2 position of a chroman ring of α-tocopherol, which is a typicalvitamin E and further binding a sugar thereto. It possesses high watersolubility and excellent resistance to oxidation. The utilization of thechromanol glycoside for the prevention and the cure of the hindrance ofthe ischemic reperfusion, however, has not been known

[0004] This invention has been initiated with a view to solving theproblems entailed by the prior art mentioned above. An object of thisinvention is to provide a novel agent for preventing and curinghindrance of ischemic reperfusion, which agent effectively acts at asmall dosage without entailing any side reaction and prevents varioushindrance induced by the ischemic reperfusion or permits the conditionof disease to be alleviated or eliminated.

[0005] Another object of this invention is to provide a novel agent forpreventing and curing hindrance of ischemic reperfusion, which agent iscapable of offering commendable resistance to oxidation and bringingeffective repression and control of free radical reactions in the partsof various internal organs affected by the hindrance of ischemicreperfusion.

[0006] Still another object of this invention is to provide a novelagent for preventing and curing hindrance of ischemic reperfusion, whichagent can be formulated as an aqueous pharmaceutical agent containing anactive component at high concentration.

DISCLOSURE OF THE INVENTION

[0007] The present inventors have performed diligent studies one afteranother in search of an agent for preventing and curing hindrance of theischemic reperfusion and, consequently, have discovered that thechromanol glycoside mentioned above is capable of dramaticallypreventing and curing morbid alterations of the hindrance of theischemic reperfusion.

[0008] Specifically, this invention concerns an agent for preventing andcuring the hindrance of the ischemic reperfusion, which agent has as anactive component thereof a chromanol glycoside represented by thefollowing general formula:

[0009] [wherein R¹, R², R³, and R⁴ independently denote a hydrogen atomor a lower alkyl groups R⁵ denotes a hydrogen atom, a lower alkyl group,or a lower acyl group, X denotes a monosaccharide residue or anoligosaccharide residue which may have a lower alkyl group or a loweracyl group substituted for the hydrogen atom of the hydroxyl group ofthe saccharide residue, n denotes an integer of 0-6, and m denotes aninteger of 1-6].

[0010] This invention also concerns the agent mentioned above, whereinsaid chromanol glycoside mentioned above is2-(α-D-glucopyranosyl)methyl-2,5,7,8-tetramethyl chroman-6-ol.

[0011] This invention further concerns the agent mentioned above,wherein said hindrance of ischemic reperfusion is a hindrance of smallintestinal mucous membrane, a hindrance of cardiac muscle ischemicreperhusion, or a hindrance of cerebral ischemic reperfusion.

[0012] This invention also concerns an agent for preventing and curingthe hindrance of ischemic reperfusion which is an aqueous pharmaceuticalagent.

BEST MODE OF EMBODYING THE INVENTION

[0013] The agent of this invention for preventing and curing thehindrance of ischemic reperfusion is characterized by having a chromanolglycoside represented by the general formula (1) mentioned above as anactive component.

[0014] In the general formula (1) mentioned above, the lower alkylgroups of R¹, R², R³, R⁴, and R⁵ are favorably to be lower alkyl groupsof carbon atoms 1-8, preferably 1-6. As concrete examples of the loweralkyl groups, methyl group, ethyl group, propyl group, isopropyl group,butyl group, isobutyl group, pentyl group, isopentyl group, hexyl group,heptyl group, and octyl group may be cited. Among other lower alkylgroups mentioned above, methyl group or ethyl group proves particularlyadvantageous. The lower acyl groups of R⁵ are favorably to be lower acylgroups of 1-8, preferably 1-6, carbon atoms. As concrete examples of thelower acyl groups, formyl group, acetyl group, propionyl group, butylylgroup, isobutylyl group, valeryl group, isovaleryl group, pivaloylgroup, hexanoyl group, heptanoyl group, and octanoyl group may be cited.Among other lower acyl groups mentioned above, acetyl group, propionylgroup, or butylyl group proves particularly advantageous. As concreteexamples of the monosaccharide residues of X, sugar residues such asglycose, galactose, fucose, xylose, mannose, rhamnose, fructose,arabinose, lyxose, ribose, allose, altrose, idose, talose, deoxyribose,2-deoxyribose, quinovose, and abequose may be cited. As concreteexamples of the oligosaccharide residues, such sugar residues asmaltose, lactose, cellobiose, raffinose, xylobiose, and sucrose whichare formed by the union of two to four such monosaccharides as mentionedabove. Among other mono saccharide residues mentioned above, glucose,galactose, fucose, xylose, rhamnose, mannose, and fructose proveparticularly advantageous. The hydrogen atom of the hydroxyl group inthe saccharide residue of X may be substituted for a lower alkyl group,preferably a lower alkyl group of 1-8 carbon atoms, or for a lower acylgroup, preferably a lower acyl group of 1-10 carbon atoms. Then, ndenotes an integer of 0-6, preferably 1-4, and m denotes an integer of1-6, preferably 1-3. As preferred examples of the chromanol glycosiderepresented by the general formula (1),2-(α-D-glucopyranosyl)methyl-2,5,7,8-tetramethyl chroman-6-ol,2-(β-D-galactopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol,2-(β-L-fucopyranosyl)methyl -2,5,7,8-tetramethylchroman-6-ol,2-(α-L-rhamnopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol,2-(β-D-xylopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol,2-(β-D-glucopyranosyl)methyl-2,5,7,8-tetramethyl chroman-6-ol,2-(β-D-fructofuranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol, and2-(α-D-mannopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol may becited.

[0015] The chromanol glycoside to be used in this invention is producedby an enzymatic reaction according to the method disclosed in theofficial gazette of JP-A-07-118,287, for example, by causing a2-substituted alcohol represented by the following general formula (2);

[0016] (wherein R¹, R², R³ ₁, R⁴ and R⁵ and n have the same meanings asdefined above) to react with an oligosaccharide, a soluble starch,starch, or cyclodextrin in the presence of an enzyme capable ofcatalyzing a corresponding transglycolytic action thereby causing aspecific hydroxyl group of sugar to be bound specifically to thehydroxyl group at the 2 position of the 2-substituted alcohol (enzymemethod).

[0017] The 2-substituted alcohol represented-by the general formula (2)used as the raw material in the reaction mentioned above (hereinafterreferred to simply as “2-substituted alcohol”) is a known substancewhich can be obtained by the method disclosed in the official gazette ofJP-B-01-43,755 or the official gazette of JP-B-01-49,135, etc. The2-substituted alcohol which satisfies the general formula (2) by havinga methyl group for each of R¹, R², R³, and R⁴, a hydrogen atom for R⁵,and 1 for n, for example, can be easily obtained as by refluxing Tororoxin diethyl ether in the presence of lithium aluminum hydride.

[0018] The enzyme which is used in the reaction mentioned above for thepurpose of catalyzing the transglycolytic action is preferred to beproperly selected to suit the kind of a sugar to be used as follows.

[0019] (1) In binding a glucose residue to the 2-substituted alcohol bythe α-bonding:

[0020] (a) The maltooligosaccharides of the range of maltose throughmaltotetrose are preferred to be acted on by an α-glucosidase, EC3.2.1.20. The α-glucosidase to be used herein does not need to bediscriminated on account of its origin. As concrete examples of theα-glucosidase, the α-glucosidase from the microorganism of genusSaccharomyces sp. made by TOYOBO Co., Ltd., the α-glucosidase from themicroorganism of genus Saccharomyces cerevisiae made by Oriental YeastIndustry Co., Ltd., the α-glucosidase from the microorganism of genusAspergillus niger made by Amano Pharmaceutical Co., Ltd., theα-glucosidase from the microorganism of genus Saccharomyces sp. made byWako Pure Chemical Industries Ltd., the α-glucosidase from themicroorganism of Bakers yeast made by SIGMA Corp., and the α-glucosidasefrom the microorganism of genus Bacillus may be cited.

[0021] (b) The soluble starch or starch is preferred to be acted on by4-α-D-glucanotransferase, EC2.4.1.25.

[0022] (2) In binding a glucose residue or a maltooligosaccharideresidue to the 2-substituted alcohol by the α-bonding:

[0023] The maltoligosaccharide, soluble starch, starch, or cyclodextrin(α, β, γ), etc. is preferred to be acted on by cyclodextringlucanotransferase, EC2.4.1.19. As typical examples of the cyclodextringlucanotransferase, the cyclodextrin glucanotransferase from themicroorganism of genus Bacillus macerans made by Amano PharmaceuticalCo., Ltd., the cyclodextrin glucanotransferase from the microorganism ofgenus Bacillus stearothermophilus made by Hayashi Genseibutsu KagakuKenkyusho, Ltd., and the cyclodextrin glucanotransferases from themicroorganisms of Bacillus megaterium and Batillus circulans ATCC 9995may be cited.

[0024] (3) In binding a glucose residue to the 2-substituted alcohol bythe β-bonding:

[0025] (a) Such oligosaccharide as cellobiose, curdlan, or laminaran,for example, which are formed by the β-bonding is preferred to be actedon by β-glucosidase, EC 3.2.1.21.

[0026] (b) Cellobiose in the presence of phosphoric acid is preferred tobe acted on by cellobiose phosphorylase, EC2.4.1.20.

[0027] (4) In binding a galactose residue to the 2-substituted alcoholby the α-bonding:

[0028] (a) Melibiose or raffinose, for example, is preferred to be actedon by α-galactosidase, EC3.2.1.22.

[0029] (5) In binding a galactose residue to the 2-substituted alcoholby the β-bonding:

[0030] (a) Lactose, for example, is preferred to be acted on byβ-galactosidase, EC3.2.1.23.

[0031] (b) Arabinogalactan, for example, is preferred to be acted on byendo-1,4-β-galactonase, EC3.2.1.89.

[0032] (6) In binding a fructose residue to the 2-substituted alcohol bythe β-bonding:

[0033] (a) Sucrose, raffinose, or melibiose , for example, is preferredto be acted on by Levansucrase, EC2.4.1.10.

[0034] (b) Sucrose, for example, is preferred to be acted on byβ-fructofuranosidase, EC3.2.1.20.

[0035] (c) Inulin, for example, is preferred to be acted on by inulinfructotransferase, EC2.4.1.93.

[0036] The conditions for the reaction mentioned above are variable withthe kind of a chromanol glycosidase and the kind of an enzyme to beused. For example, when the chromanol glycoside which satisfies thegeneral formula (1) by having 1 for m is to be synthesized by using anα-glucosidase, the 2-substituted alcohol is preferred to be dissolved inthe sugar solution. Accordingly, it is preffered to add an organicsolvent to the solution. As concrete examples of the organic solvent,dimethylsulfoxide, N,N-dimethylformamide, methanol, ethanol, acetone,and acetonitrile may be cited. Among other organic solvents,dimethylsulfoxide and N,N-dimethylformamide prove particularly suitablein consideration of the ability to improve the transfer activity of theα-glucosidase. The concentration of the added organic solvent is in therange of 1-50 (v/v) %. In view of the efficiency of the reaction, it ispreferred to be in the range of 5-35 (v/v) %.

[0037] The 2-substituted alcohol is preferred to have a saturatedconcentration or a concentration approximating closely thereto in thereaction solution. The kind of sugar to be used is preferred to be sugarhaving low molecular weight, in the rang of approximately from maltosethrough maltotetraose. The maltose is a preferred choice. Theconcentration of the sugar is in the range of 1-70 (w/v) % preferably30-60 (w/v) %. The pH is in the range of 4.5-7.5, preferably 5.0-6.5.The reaction temperature is in the range of 10-70° C., preferably 30-60°C. The reaction time is in the range of 1-40 hours, preferably 2-24hours. Needless to mention, these conditions are affected by the amountof the enzyme to be used, for example. After the reaction is completed,the chromanol glycoside aimed at is obtained at high purity by treatingthe reaction solution by the column chromatography using XAD (producedby Japan Organo Co., Ltd.) as a carrier.

[0038] When the chromanol glycoside satisfying the general formula (1)by having 1 for m is to be synthesized by using cyclodextringlucanotransferase, the reaction condition is preferable that the2-substituted alcohol is dissolved in the sugar solution. It ispreferable to add organic solvent, such as dimethyl sulfoxide,N,N-dimethyl formamide, methanol, ethanol, acetone, acetonirtile, etc..The concentration of the added organic solvent is in the range of 1-50(v/v) %. In view of the efficiency of the reaction, this concentrationis preferred to be in the range of 5-35 (v/v) %. The 2-substitutedalcohol is preferred to have a saturated concentration or aconcentration approximating closely thereto in the reaction solution.

[0039] As preferred concrete examples of the sugar to be used in thereaction mentioned above, maltoligosaccharide having polymerizationdegree exceeding that of maltotriose, soluble starch, starch, andcyclodextrin (α, β, γ) may be cited. The concentration of the sugar isin the range of 1-70 (w/v) %, preferably 5-50 (w/v) %. The pH is in therange of 4.5-8.5, preferably 5.0-7.5. The reaction temperature is in therange of 10-70° C., preferably 30-60° C. The reaction time is in therange of 1-60 hours, preferably 2-50 hours. These conditions, however,are affected by the amount of the emzyme to be used. The chromanolglycoside which has been obtained by the reaction described above formsa mixture satisfying the general formula (1) by having approximately 1through 8 for m, Then, by treating this mixture with glucoamylase(EC3.2.1.3), the chromanol glycoside satisfying the general formula (1)by having 1 for m can be obtained exclusively. In this case, thereaction temperature is in the range of 20-70° C., preferably 30-60° C.and the reaction time is in the range of 0.1-40 hours, preferably 1-24hours. However, these conditions are affected by the amount of theenzyme to be used. Then, by subjecting the solution remaining after thetreatment with the glucoamylase mentioned above to column chromatographyusing XAD (made by Japan Organo Co., Ltd.) as a carrier, the chromanolglycoside satisfying the general formula (1) by having 1 for m isobtained at high purity.

[0040] The chromanol glycoside satisfying the general formula (1) byhaving 1 or 2 for m is exclusively obtained by causing the chroanolglycoside in the form of a mixture satisfying the general formula (1) ofaporoximately 1 through 8 for m obtained by cyclodextringlucanotransferase to be acted on by β-amylase (EC3.2.1.2). In thiscase, the reaction temperature is in the range of 20-70° C., preferably30-60° C. and the reaction time is in the range of 0.1-40 hours,preferably 1-24 hours. These conditions, however, are affected by theamount of the enzyme to be used. When the solution remaining after thetreatment with the β-amylase is treated by the column chromatographyusing the XAD (made by Japan Organo Co. ,Ltd.) as a carrier, thechromanol glycoside satisfying the general formula (1) by having 2 formis obtained at high purity and, at the same time, the chromanolglycoside satisfying the general formula (1) by having 1 for m isobtained.

[0041] In obtaining the chromanol glycoside satisfying the generalformula (1) by having not less than 3 for m, the chromanoll glycoside ofhigh purity can be obtained for each value of m by preparing chromanolglycoside in the form of a mixture satisfying the general formula (1)having approximately 1 through 8 for m with cyclodextringlucanotransferase and treating this chromanol glycoside, for example,by the fractionation chromatography using HPCL, etc.

[0042] The mode of embodiment, as described above, consists in binding aglucose residue or a maltoligosaccharide residue as a sugar residue tothe 2-substituted alcohol. The mode of embodiment also can be likewiseused advantageously in this invention, in which a galactose residue, aβ-glucose residue, a mannose residue, or a fructose residue as a sugarresidue is bound to the 2-substituted alcohol. In this mode, thechromanol glycoside aimed at is obtained at high purity by following thesame procedure as the mode of embodiment mentioned above while suitablyselecting a proper enzyme among the enzymes described in the foregoingparagraph concerning the enzymes capable of catalyzing thetransglycolysis mentioned above (the official gazette ofJP-A-09-249,688, Japanese Patent Application No. 9-176,174).

[0043] The chromanol glycoside to be used in this invention can beproduced alternatively by preparing the 2-substituted alcohol mentionedabove in a form having the hydroxyl group at the 6 position thereofprotected with a protecting group (hereinafter referred to as “sugaracceptor”) and a sugar derivative having a leaving group introduced tothe anomer position and having the other hydroxyl group protected with aprotecting group (hereinafter referred to as “sugar donor”) andsubjecting the sugar acceptor and the sugar donor to a condensingreaction in accordance with the method disclosed in the official gazetteof Japanese Patent Application No. 10-75,599 (organic synthesis method).

[0044] As concrete examples of the protecting group for protecting thehydroxyl group at the 6 position of the sugar acceptor to be used in thereaction mentioned above, acetyl group, benzoyl group, pivaloyl group,chloroacetyl group, levulinoyl group, benzyl group, p-methoxybenzylgroup, allyl group, t-butyldimethylsilyl group, t-butyldiphenylsilylgroup, trimethylsilyl group, and trityl group may be cited. Among otherprotecting groups mentioned above, acetyl group and benzoyl group proveparticularly advantageous.

[0045] As concrete examples of the leaving group introduced to theanomer position of the sugar donor to be used in the reaction mentionedabove, halogen atoms such as chlorine, bromine, and fluorine, sulfurcompounds such as thiomethyl group, thioethyl group, and thiophenylgroup, and trichloroacetoimide group may be cited. Among other leavinggroups mentioned above, bromine, chlorine, thiomethyl group, thioethylgroup, thioplhenyl group, and trichloroacetoimide group proveparticularly advantageous. As concrete examples of the protecting groupfor protecting the hydroxyl group at a position other than the anomerposition, acyl type protecting groups such as acetyl group, benzoylgroup, pivaloyl group, chloroacetyl group, and levulinoyl group andether type protecting groups such as benzyl group, p-methoxybenzylgroup, allyl group, t-butyldimethylsilyl group, t-butyldiphenylsilylgroup, trimethylsilyl group, and trityl group may be cited. Among otherprotecting groups mentioned above, acyl type protecting groups,especially acetyl group prove particularly advantageous.

[0046] The sugar donor can be easily prepared by introducing protectinggroups to all the hydroxyl groups of a given sugar and then substitutingthe protecting group at the anomer position for a leaving group inaccordance with the known method.

[0047] To illustrate the condensing reaction of the sugar acceptor andthe sugar donors the first step is to dissolve the sugar acceptor andthe sugar donor in a nonpolar solvent. The amounts of the sugar acceptorand the sugar donor to be charged are expected to be such that the molarratio of the sugar donor to the sugar acceptor fall in the range of1.0-1.5, preferably 1.1-1.3. As concrete examples of the nonpolarsolvent, methylene chloride and benzene may be cited.

[0048] Then, the condensing reaction of the sugar donor and the sugaracceptor is carried out under an anhydrous condition in the presence ofan activating agent. As concrete examples of the activating agent, borontrifluoride ether complex, silver perchlorate, silver trifluoromethanesulfonate, mercury bromide, mercury cyanide,N-iodosuccinicimide-trifluoromethanesulfonate,dimethylmethylthiosulfonium triflate, and p-toluenesulfonic acid may becited. Particularly when bromine is used as a leaving group for thesugar derivative, it is commendable to use such a heavy metal salt assilver perchlorate. The reaction temperature is in the range of 5-30°C., preferably 10-25° C., and the reaction time is in the range of 12-48hours, preferably 20-30 hours.

[0049] Subsequently, 2-(β-L-fucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol,2-(α-L-rhamnopyrasyl)methyl-2,5,7,8-tetramethylchroman-6-ol,2-(β-D-xylopyranosyl) methyl-2,5,7,8-tetramethyl chroman-6-ol, etc. canbe obtained by purifying the consequently obtained reaction product, forexample, by the silica gel column chromatography thereby depriving theprotecting group of its function with sodium hydroxide and amethanolichydrochloric acid, for example (JP-A-10-75,599).

[0050] The chromanol glycoside which is obtained by the enzyme techniqueor the technique of organic synthesis generally is an amphipaticmolecule possessing exceptionally high solubility in water (about 100g/100 ml) and abounding in solubility oil (octanol/water typedistribution coefficient>3). That is, the chromanol glycoside accordingto this invention may well be called a water-soluble vitamin E endowedwith high affinity for oil. Unlike the conventional vitamin E derivativewhich is insoluble or poor soluble in water, the chromanol glycosideaccording to the present invention retains high affinity for oil evenwhen it is used as dissolved in water and, therefore, possesses anability to permeate cell membranes and further enter cell interiors,fortifies the in vivo protecting system in resisting oxidation, preventsthe hindrance of ischemic reperfusion by effectively repressing andcontrolling the active oxygen and the free radical in the parts affectedby the hindrance of ischemic reperfusion, or brings a prominentimprovement capable of healing the condition of the disease due to thehindrance of the ischemic reperfusion. Further, the chromanol glycosidewhich is obtained by the reaction mentioned above extremely excelstocopherol, Tororox, or 2-substituted alcohol in terms of thermalstability and pH stability.

[0051] The agent of this invention for preventing and curing thehindrance of ischemic reperfusion can be orally or otherwiseadministered to patients in the form of a composition having thechromanol glycoside mentioned above formulated with pharmaceuticallyallowable carriers or dissolved or suspended in a pharmaceuticallyallowable solvent.

[0052] For the purpose of applying the agent to oral administration, thechromanol glycoside mentioned above is suitably mixed with properadditives such as, for example, milk sugar, sucrose, mannitol, cornstarch, synthetic or natural rubber, and crystalline cellulose, bindingagents such as, for example, starch, cellulose derivatives, gum arabic,gelatine, and polyvinyl pyrrolidone, decaying agents such as, forexample, carboxymethyl cellulose calcium, carboxymethyl cellulosesodium, starch, corn starch, and sodium alginate, glossing agents suchas, for example, talc, magnesium stearate, and sodium stearate, andfillers or diluents such as, for example, calcium carbonate, sodiumcarbonate, calcium phosphate, and sodium phosphate and formulated assolid pharmaceutical agents as tablets, dust (powder), pills, andgranules. It may be otherwise encapsulated with hard or soft gelatinecapsules. These solid pharmaceutical agents maybe furnished with anenteric coating using any of the coating bases such as, for example,hydroxypropylmethyl cellulose phthalate, hydroxypropylmetyl clluloseacetate succinate, cellulose acetate phthalate, and methacrylatecopolymers. Further, the chromanol glycoside mentioned above may beformulated as liquid preparations such as syrup agents and elixir agentsby dissolving this glycoside in an inert dilutent popularly used inpurified water and, as occasion demands, suitably adding to the solutiona wetting agent, an emulsifier, a dispersion auxiliary, a surfactant, anedulcorant, a flavor, an aromatic substance, or the like.

[0053] For the administration not via the mouth of the agent of thisinvention for preventing and curing the hindrance of ischemicreperfusion, the chromanol glycoside mentioned above may be formulatedin the form of a sterilized aqueous solution, a non-aqueous solution, asuspension, a ribosome, or an emulsion, preferably an injection gradesolution or a spray grade sterilized aqueous solution, as suitablycombined with purified water, a proper buffer solution such as aphosphate buffer solution, a physiological common salt solution, aphysiological salt solution such as a Ringer's solution or a Locke'ssolution, ethanol, glycerin, and a popular surfactant. Such apreparation may be administered intravenously, hypodermically,intramuscularly, intra-abdominally, intestinally, and intrabronchially.The liquid preparation in this case is preferred to have a physiologicalpH, preferably a pH in the range of 6-8. Further, the agent of thisinvention for preventing and curing the hindrance of ischemicreperfusion may be administered as embedded by a pellet or as preparedin the form of a suppository using a suppository basis.

[0054] In the preparations and the modes of administration mentionedabove, those which are appropriate for a given patient are selected by aphysician in charge.

[0055] Though the concentration in which the chromanol glycoside iscontained in the agent of this invention for preventing and curing thehindrance of ischemic reperfusion is varied by the mode ofadministration, the kind of disease, the degree of seriousness ofdisease, and the dosage aimed at, it is generally in the range of0.1-100 wt. %, preferably 1-90 wt. %, based on the total weight of theraw materials. Particularly when the preparation of this invention isorally administered, the concentration is in the range of 1-100 wt. %,preferably 5-90 wt. %, based on the total weight of the raw materials.In the case of the administration parenterally, the concentration is inthe range of 0.1-90 volume %, preferably 1-80 volume %, based on thetotal volume of the raw materials. In this case, if the concentration ofthe chromanol glycoside exceeds the upper limit of the range mentionedabove, the excess will not bring a proportionate addition to the effectin alleviation of the condition of disease. Conversely, if thisconcentration is less than the lower limit of the range, it will be at adisadvantage in preventing the alleviation of the condition of diseasefrom being fully achieved.

[0056] The aforementioned dosage of the agent of this invention forpreventing and curing the hindrance of ischemic reperfusion is variablewith the age, body weight, and symptom of a relevant patient, the modeand method of administration aimed at, the effect of treatment, and theduration of treatment. The accurate dosage is to be decided by aphysician. Generally when the preparation is orally administered, theamount of the preparation as reduced to the dosage of the chromanolglycoside is in the range of 0.1-10000 mg/kg of body weight/day, onethrough three times daily. In this case, when the daily oral dosage islarge, the preparation may be administered at a rate of a plurality oftablets in one dosage. When the agent of this invention for prevntingand curing the hindrance of ischemic reperfusion is administeredparenterally, the preparation is administered once wholly or one orthree times as split daily in such an amount that the dosage ofchromanol glycoside as reduced to the dosage of the chromanol glycosidewill be in the range of 0.01-1000 mg/kg of body weight/day.

[0057] The agent of this invention for prevnting and curing thehindrance of ischemic reperfusion can be utilized for preventing andcuring the hindrances caused in various sites such as, for example,heart, stomach, small intestine, liver, spleen, kidney, brain, eyeball,and skin by ischemic reperfusion and various hindrances caused duringthe transplantation of an internal organ. To be specific, the hindrancesmentioned above include such hindrances as hindrances of cerebralischemic reperfusion and cardiac muscular ischemic reperfusion, thehindrances which arise during the reconstruction of ranial bloodcirculation in various infarctions such as cerebral infarction,myocardial infarction, and pulmonary infarction, during the formation ofcardiopulmonary bypass, and during the transplantation of an internalorgan, and the hindrances of microcirculation in the mucous membrane ofthe digestive system and the surface of the eyeball caused by stress,for example. The hindrances of the cerebral ischemic reperfusionmentioned above include ischemic cerebral edema and likes and thehindrances of the myocardial ischemic reperfusion include ventriculararrhythmia observed before and after elimination of coronary spasm andresumption of blood circulation by solution of thrombus, heart rupturedue to infarct tissue internal hemorrhage, and imperfect recuperation ofthe heart function and stunned myocardium after the fallaway ofmechanical heart-lung encountered during the surgical operation of theheart.

[0058] Pharmacologic Test

[0059] The pharmacological effect of the agent of this invention forpreventing and curing the hindrance of ischemic reperfusion will bedescribed more specifically below with reference to pharmaceutical testsusing animals.

[0060] In each pharmacological test, the2-(α-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol (TMG)represented by the following formula (3) which was produced by themethod described in Example 1 cited in the official gazette ofJP-A-07-118,287was used as a chromanol glycoside.

[0061] [Repressing Effect of Morbid Alteration with Model of Hindranceof Small Intestinal Ischemic Reperfusion]

[0062] A model of hindrance of small intestinal ischemic reperfusion wasproduced in a rat by ligating the celiacarteria and at the same timeblocking the superior mesenteric artery with a clip to suspend thebloodstream therethrough for 30 minutes, then relieving them of therespective obstructions and allowing reperfusion to last for 60 minutes,and thereafter rating the hindrance of mucous membrane in the smallintestine. When a morbid alteration occurred in the mucous membrane ofthe small intestine, hemorrhage in the small intestinal lumen, proteintransudation, and increase of the thiobarbituric acid (TBA) reactionsubstance (index of lipid peroxidation) in the mucous membrane wererecognized. With such substances as the index, the model was tested forthe effect of the chromanol glycoside in suppressing the morbidalteration of the hindrance of ischemic reperfusion.

[0063] SD type male rats (body weights 180-200 g) divided into groups ofsix heads were made to keep 18 hours' fasting before they were put touse in the test. They were subjected to median ventrotomy underanesthesia with urethane (1000 mg/kg) and caused to assume a state ofischemia by ligating the celiac arteria and then blocked the superiormesenteric artery at the root with a clip. After the elapse of 30minutes thence, a TMG preparation obtained by thoroughly dissolving TMGat a concentration of 0.8 mg/ml in physiological common salt solutionwas administered to the rats through intravenous injection at a rate of4 mg/kg of body weight, and then reperfusion was started by removing theclip. After 60 minutes of the reperfusion, the rats were sacrificed byreleasing blood from the aorta. The small intestines were excised fromthe sacrificed rats and the hemoglobin transudated from the smallintestinal lumen, the transudated protein, and the TBA reactionsubstance in the small intestinal mucous membrane were determinedquantitatively.

[0064] The results consequently obtained are shown in Table 1 togetherwith the results obtained of the normal group of rats which had notundergone the treatment of ischemic reperfusion and the administrationof TMG preparation and the results obtained of the control group of ratswhich had undergone the administration by intravenous injection ofphysiological common salt solution in the same amount in the place ofthe TMG preparation after the ischemia and before the reperfusion. Theitems of the rating mentioned above were determined by the followingmethods.

[0065] (1) Method for Measurement of Hemoglobin Transudated from theSmall Intestinal Lumen and Transudated Protein

[0066] As the index of the hindrance of the small intestinal mucousmembrane by ischemic reperfusion, the amount of the hemoglobin and theamount of protein transudated into the small intestinal lumen weremeasured. A 30-cm adoral portion of the small intestine was excised froma site at a distance of 5 cm from the terminal end of the ileum, theinterior of the small intestinal lumen was washed from the adoral sidewith 10 ml of a cold physiological common salt solution, and the washingrecovered consequently was put to use for the measurement. The amount ofthe hemoglobin transudated was measured by the cyanmethemoglobintechnique (Cannan, R. K.: Am. J. Clin. Path., 44, 207-210, 1965) using ahemoglobin measuring kit (made by Wako Pure Chemical Industries, Ltd.And sold under the trademark designation of “Hemoglobin Test Wako”).Specifically, 0.2 ml of the intestine washing solution was intimatelymixed with 5 ml of a coloring reagent (0.78 mM potassium cyanide+0.61 mMpotassium ferricyanide). The produced mixture was left standing at roomtemperature for 5 minutes and then tested for absorbance with aspectrophotometer (made by Jasco Engineering K.K. and sold under thetrademark designation of “JASCO Ubest-30, UV/VIS Spectrophotometer”)at awavelength of 540 nm. The hemoglobin content of the washing wascalculated from the calibration curve obtained in advance with thecyanmethemoglobin standard solution (18 g of hemoglobin/dl+3.1 mMpotassium cyanide+0.61 mM potassium ferricyanide) and was reported atthe amount of hemoglobin per unit length of the small intestine. Theamount of the protein transudated was measured by the Lowry technique(Lowry, O. H.: J. Biol. Chem., 193, 265-275, 1951) using a proteinmeasuring kit (made by Sigma Chemical Co., Ltd.).

[0067] (2) Method for Measurement of TBA Reaction Substance in SmallIntestinal Mucous Membrane

[0068] This measurement was carried out by the Ohkawa technique (Ohkawa,H.: Anal. Biochem., 95, 351-358, 1979). The adoral 10-cm portion of thesmall intestine was excised from the site at a distance of 5 cm from theterminal end of the ileum, cut open in the direction of major axis, andstripped of the mucous membrane with the aid of two slide glasses. Themembrane was diluted to 10 times its own weight (wt. %) with an aqueous10 mM phosphate buffer-30 mM potassium chloride solution as homogenized(by means of a homogenizer fitted with a Pyrex homogenizer grade glass10 ml in volume, made by Iuchi Seiei Do K.K., and operated at 100 rpm).The portion, 0.2 ml, of the homogenate, and 0.6 ml of distilled waterand 0.2 ml of an aqueous 8.1% sodium disulfate solution added thereto,and 1.5 ml of a 20% phosphate buffer solution of pH 3.5, 1.5 ml of 0.8%TBA, and 40 μl of 1% BTH further added thereto were heated altogether inan oil bath (made by Taitekku K.K. and sold under the trademarkdesignation of “OH-50P”) at 95° C. for one hour and then cooled for 10minutes. The produced mixture and 1.0 ml of distilled water and 5.0 mlof butanol pyridine (butanol:pyridine=15:1, v/v) added thereto werestirred and then centrifuged (1500 g, 10 minutes) at room temperature.The supernatant consequently obtained was tested for absorbance by meansof a spectrophotometer (made by Jasco Engineering K.K. and sold underthe trademark designation of “JASCO Ubset-30, UV/VIS Spectrophtometer)at a wavelength of 535 nm. From the blank using 0.8 ml of distilledwater and the standard using 0.3 ml of distilled water and 0.5 ml of TEPrespectively in the place of 0.2 ml of the homogenate of smallintestinal mucous membrane and 0.6 ml of distilled water, a calibrationcurve was obtained. The content of the TBA reaction substance in a givensample was calculated with reference to this calibration curve. TABLE 1Hemoglobin Protein TBA reaction transudated in transudated in substancein small small small intestinal mucous intestinal intestinal membranelumen lumen (nmol/g on wet (mg/cm) (μg/cm) basis) Normal group 0.5 ± 0.40.3 ± 0.1 57.1 ± 9.3  Control group 3.4 ± 1.5 1.6 ± 0.3 285.6 ± 105.7TMB  0.4 ± 0.3**  0.5 ± 0.1** 112.8 ± 33.6* administration group (4mg/kg of body weight), i.v.

[0069] It is clearly noted from Table 1 that the model of hindrance ofischemic reperfusion (control group) showed discernible increases inhemorrhage and protein transudation in the small intestinal lumen and inthe TBA reaction substance in the mucous membrane and the TMGadministration group showed significant repressions in these values.This fact indicates that the agent of this invention for preventing andcuring the hindrance in ischemic reperfusion brought prominentrepression of the hindrance of small intestinal mucous membrane inducedby the iscmetic reperfusion.

[0070] [Effect of Repressing Morbid Alteration by Model of Hindrance ofIschemic Reperfusion]

[0071] A model of hindrance of ischemic reperfusion in the cardiacmuscle was produced by performing perfect ischemia for 30 minutes andsubsequently performing reperfusion for 20 minutes on the sample of theperfused heart excited from a rat (Langendorff sample). It is known thatthis model establishes a bell-shaped curve between the decline of theheart function, arrhythmia of the reperfusion, particularly thefrequency of appearance and the duration of the ventricular fibrillationand the duration of the preceding ischemia and manifests the ventricularfibrillation at a frequency of 100% during the reperfusion after 30minutes' ischemia (Okabe et al,. Eur. J. Pharmacol., 248: 33, 1993). Theeffect of the chromanol glycoside in repressing the morbid alteration ofthe hindrance of the ischemic reperfusion in the cardiac muscle wasstudied by using the model mentioned above, with various parameters ofthe function of the heart and the manifestation of arrhythmia asindexes.

[0072] SD type male rats (having body weights 190-210 g) which hadundergone a heparin treatment (200 units, i.v.) were fixed on a dorsalposition and subjected to laparotomy under anesthesia with diethylether. Each of the rats was cut open in the chest along the median linewith the diaphragm sectioned along the lower edge of the rib before theheart was quickly excised. The heart was placed in a petri dish filledin advance with a perfusion liquid and stripped of the tissue enclosingthe periphery thereof. The Langendorff sample (Langendorff: PflugersArch., 61: 2931 1895) was made of this heart by inserting a cannule intothe aorta. Immediately after the production of this sample, theperfusion was initiated reversely via the cannula with the pressure setat 100 cm H₂O. The perfusion liquid, namely a modified Krebs-Henseleitsolution which was composed of 118 MM of sodium chloride, 4.7 mM ofpotassium chloride, 2.5 mM of calcium chloride, 1.2 mM of magnesiumsulfate, 1.2 mM of potassium dihydrogen phosphate, 25 mM of sodiumhydrogen carbonate, and 11 mM of glucose, was used as aerated with amixed gas consisting of 95% oxygen and 5% carbon dioxide.

[0073] A balloon made of latex and filled in advance with distilledwater was inserted into the left ventricle and connected to a pressuretransducer (“TP-400T”, Nippon Kogen K.K., Tokyo). The maximum leftventricular pressure (LVDP) was determined through the medium of anamplifier for pressure determination (made by Nippon Koden K.K. and soldunder the product code of “AP-621G”) and the primary differential valueof the left ventricular pressure (LVdP/dt_(max)) through the medium of adiffernetial computing unit (made by Nippon Koden K.K. and sold underthe product code of “EQ-621G”). The heart rate (HR) was determined bydriving a cardiograph (made by Nippon Koden K.K. and sold under theproduct code of “AT-601G”) with the pulse wave of the left ventricularpressure. The amount of the perfusion liquid discharged from thecoronary sinus was measured as the amount of coronary perfusion (CF) bythe use of a stalagmometer (made by Technical Supply K.K. and sold underthe product code of “DCBF-1”).

[0074] An electrocardiogram was recorded through by two platinumelectrodes set to a sample (Okabe et al.: Eur. J. Pharmacol., 248: 33,1993). The protocol of the experiment on ischemic reperfusion wasperformed with the reperfusion performed for 20 minutes after thecomplete ischemia continued for 30 minutes and the manifestation of thearrhythmia of reperfusion was raged and sorted in accordance with theguide line proposed by Lambeth Conventions (Walker et al.: Cardiovasc.Res., 22: 447, 1988) with the necessary modifications. That is,theventricular tachycardia (VT) was taken as not less than four successivepremature contractions (PVC) and the ventricular fibrillation (VF) wastaken as the waveform not discriminable by QRS. Then, the cardiacstandstill (CA) was taken as the case wherein the electrocardiogramassumed a flat waveform and the latent time thereof was taken as having600 seconds as its maximum.

[0075] The perfusion was continued for about 10 minutes and leftstabilizing, then suspended for 30 minutes by way of a treatment forischemia as mentioned above, and thereafter quickly resumed and placedunder observation for 20 minutes. The TMG was advanced through achemical injection path disposed halfway in the length of the aortacannula and injected into the cannula for five minutes prior to thetreatment or for 10 minutes immediately after the resumption of theperfusion. The rate of injection of the TMG was adjusted with a syringepump (“Model-901”, Harvard Apparatus, U.S.A.) so as to equal about1/10,000 of the amount of coronary perfusion.

[0076] The TMG was dissolved in the modified Krebs-Henseleit solution soas to assume a final concentration in the range of 1-10 μM. In thiscase, the pH was stable at 7.4 and the osmotic pressure was in the rangeof 290-325 mosmols.

[0077] The action of the 5-minute treatment with the TMG on the functionof the heart during the normal perfusion is shown in Table 2. Further,the effect of the TMG treatment performed for 5 minutes prior to theischemia on the decline of the function of the heart due to thehindrance of ischemic reperfusion observed in the samples of perfusiontaken from rats is shown in Table 3, the effect of the TMG treatmentperformed for 1.0 minutes immediately after start of the reperfusion inTable 4, and the effect of the TMG treatment performed for five minutesprior to the ischemia and for 10 minutes immediately after start of thereperfusion relative to the manifestation of the arrhythmia ofreperfusion in Table 5 respectively. The rats to which the modifiedKrebs-Henseleit solution was exclusively injected via the chemicalinjection path were taken as the control group. TABLE 2 TMGconcentration 5 μM 10 μM Before After Before After treatment treatmenttreatment treatment LVDP 90.6 ± 3.8 88.9 ± 5.6 85.9 ± 4.6 82.8 ± 6.0(mmHg) LVdP/dt_(max) 1986 ± 121 2048 ± 113 2128 ± 156 2246 ± 163(mmHg/s) LVEDP 11.4 ± 1.2  9.6 ± 0.8 12.2 ± 1.5 11.1 ± 1.7 (mmHg) CF 19.1 ± 1.5 20.9 ± 2.0 11.4 ± 1.9 13.2 ± 2.1 (ml/min) HR 316 ± 24 291 ±16 258 ± 18 263 ± 22 (beats/min)

[0078] TABLE 3 Time elapsed after start of reperfusion −30 minutes −20minutes −10 minutes 0 minute 5 minutes 10 minutes 15 minutes 20 minutesLVDP Control — — — — 2.4 ± 5.9 3.8 ± 6.9 7.1 ± 4.3 11.8 ± 7.9 (% TMG 1μM — — — — 30.2 ± 4.8** 40.6 ± 6.6** 58.9 ± 9.1** 63.8 ± 7.0**recovery)^(a) TMG 5 μM — — — — 32.4 ± 3.9** 59.3 ± 6.6** 70.8 ± 10.1**78.8 ± 11.8** TMG 10 μM — — — — 80.6 ± 4.8** 102.8 ± 7.8** 98.8 ± 10.4**101.8 ± 15.6** LVdP/ Control — — — — 4.8 ± 8.9 8.4 ± 4.8 12.9 ± 8.6 20.9± 11.8 dt_(max) TMG 1 μM — — — — 46.8 ± 10.3** 49.1 ± 5.3** 58.2 ±12.9** 63.2 ± 13.3** (% TMG 5 μM — — — — 62.8 ± 8.9** 70.9 ± 10.4** 79.9± 13.8** 90.1 ± 15.5** recovery)^(a) TMG 10 μM — — — — 91.4 ± 9.9**113.3 ± 8.6** 103.9 ± 11.1** 99.8 ± 13.4** LVEDP Control 12.4 ± 2.6 11.8± 3.2 28.3 ± 2.9 38.8 ± 5.4 59.8 ± 6.4 50.2 ± 3.0 40.9 ± 5.9 35.3 ± 7.5(mmHg) TMG 1 μM 14.4 ± 5.9 12.1 ± 4.0 20.1 ± 3.1 38.0 ± 8.9 41.2 ± 4.8*33.4 ± 4.0** 30.1 ± 2.0** 25.0 ± 1.9** TMG 5 μM 11.8 ± 3.8 10.9 ± 6.119.9 ± 7.1 22.7 ± 4.6* 32.8 ± 7.3** 20.4 ± 4.8** 15.8 ± 2.6** 12.2 ±3.4** TMG 10 μM 13.8 ± 4.3 12.5 ± 5.4 15.8 ± 3.8* 18.6 ± 5.9** 22.6 ±3.2** 20.8 ± 4.4** 13.9 ± 7.4** 11.8 ± 3.9** CF Control — — — — 34.6 ±1.8 41.8 ± 3.0 40.1 ± 4.2 42.8 ± 3.2 (% TMG 1 μM — — — — 68.3 ± 2.6**79.0 ± 1.5** 80.3 ± 3.9** 80.0 ± 1.2** recovery)^(a) TMG 5 μM — — — —81.9 ± 4.6** 90.8 ± 2.3** 91.2 ± 3.4** 90.6 ± 3.0** TMG 10 μM — — — —110.2 ± 10.1** 104.5 ± 11.0** 99.2 ± 8.8** 108.2 ± 7.6** HR Control — —— — 32 ± 20 104 ± 18 321 ± 21 297 ± 41 (beats/ TMG 1 μM — — — — 41 ± 4092 ± 21 290 ± 22 288 ± 30 min) TMG 5 μM — — — — 194 ± 50** 288 ± 38**289 ± 30 280 ± 35 TMG 10 μM — — — — 268 ± 38** 304 ± 42** 310 ± 38 309 ±31

[0079] TABLE 4 Time elapsed after start of reperfusion −30 minutes −20minutes −10 minutes 0 minute 5 minutes 10 minutes 15 minutes 20 minutesLVDP Control — — — — 3.8 ± 4.0 2.9 ± 5.2 7.6 ± 6.3 10.8 ± 5.6 (% TMG 5μM — — — — 4.9 ± 5.2 4.8 ± 7.2 5.6 ± 7.0 6.5 ± 8.1 recovery)^(a) TMG 10μM — — — — 10.8 ± 5.5* 12.8 ± 3.1* 34.6 ± 4.8** 52.6 ± 7.8**LVdP/dt_(max) Control — — — — 9.6 ± 8.2 12.3 ± 5.9 12.9 ± 8.1 25.4 ±11.3 (% TMG 5 μM — — — — 12.8 ± 5.6 15.8 ± 10.1 21.4 ± 9.2 38.8 ± 8.4recovery)^(a) TMG 10 μM — — — — 19.6 ± 4.2* 38.8 ± 3.6** 44.8 ± 5.1**50.9 ± 3.0** LVEDP Control 18.8 ± 3.4 19.0 ± 5.5 29.9 ± 4.3 40.4 ± 7.063.2 ± 8.0 55.5 ± 3.3 40.1 ± 4.3 31.1 ± 8.0 (mmHg) TMG 5 μM 15.5 ± 5.618.2 ± 3.8 32.6 ± 8.4 52.1 ± 6.3 64.9 ± 12.6 43.9 ± 7.0 35.9 ± 6.5 30.2± 4.8 TMG 10 μM 20.6 ± 2.4 12.8 ± 7.6 36.2 ± 8.3 39.0 ± 10.1 45.8 ± 7.1*40.9 ± 3.2** 30.0 ± 4.2** 19.8 ± 2.8** CF Control — — — — 42.8 ± 2.443.3 ± 3.8 43.9 ± 4.2 45.5 ± 2.9 (% TMG 5 μM — — — — 50.5 ± 5.6 49.2 ±5.3 51.8 ± 7.2 55.9 ± 8.0 recovery)^(a) TMG 10 μM — — — — 72.8 ± 3.0**80.0 ± 2.9** 79.0 ± 7.8** 83.4 ± 4.2** HR Control 51 ± 36 96 ± 53 278 ±46 321 ± 41 (beats/min) TMG 5 μM — — — — 42 ± 41 118 ± 36 312 ± 38 338 ±54 TMG 10 μM 78 ± 21** 188 ± 43** 298 ± 63 286 ± 41

[0080] TABLE 5 1 μM TMG administration group 5 μM TMG administrationgroup 10 μM TMG administration group For 5 minutes For 10 minutes For 5minutes For 10 minutes For 5 minutes For 10 minutes Control prior toafter start of prior to after start of prior to after start of groupischemia reperfusion ischemia reperfusion ischemia reperfusion FrequencyPVC 4/4 — — 2/4 4/4 2/4 3/4 of VT 4/4 — — 3/4 4/4 3/4 4/4 occurrence VF4/4 — — 1/4 3/4 1/4 2/4 CA 3/4 — — 1/4 2/4 1/4 1/4 Duration Arrhythmia16.3 ± 3.4(4) 11.4 ± 1.8(3)* 15.8 ± 5.6(4) 7.6 ± 2.9(3)* 13.0 ± 1.2(4)6.8 ± 1.6(3)** 9.6 ± 1.9(4)* (minutes) Normal 4.2 ± 2.8(4) 10.6 ±1.3(4)* 5.0 ± 3.2(4) 12.4 ± 5.4(4)* 5.8 ± 2.6(4) 12.9 ± 2.4(4)* 8.8 ±2.6(4)* sinus rhythm

[0081] As clearly noted from Table 2 that the administration of TMG hasbeen confirmed to bring no adverse effect on any of the largest leftventricular pressure (LVDP), the left ventricular pressure primarydifferential value (LVdP/dt_(max)), the left ventricular extension finalpressure (LVEDP), the amount of coronary perfusion (CF), and the heartrate (HR).

[0082] It is clearly noted from Table 3 and Table 4 that in the TMGadministration group, the declines in LVDP, LVdP/dt_(max), and CFobserved after the reperfusion were improved depending on theconcentration and the rise in LVEDP was repressed depending on theconcentration. While the HR showed a conspicuous decrease during theinitial stage of the reperfusion (5-10 minutes after the start of thereperfusion), this decrease was repressed in the TMG administrationgroup.

[0083] It is further clear from Table 5 that the TMG, in either thetreatment prior to ischemia or the treatment immediately after thereperfusion, decreased the frequency of the manifestation of arrhythmia,significantly shortened the duration of the arrhythmia observed duringthe reperfusion, and significantly elongated the contraction of thenormal sinum rhythm.

[0084] [Effect of Repressing Morbid Alteration by Model of Hindrance inCerebral Ischemic Reperfusion]

[0085] A model rat for the hindrance of cerebral ischemic reperfusionwas produced by using a four blood vessel occlusion model rat (CranialNerve, Vol. 47, pp. 369-375, 1995) as the model of the prosencephalonischemic reperfusion and performing on the rat the cerebral ischemia forfive minutes and then continuing the reperfusion for 120 minutes. As aresult of the ischemic reperfusion, the cerebral edema, i.e. a morbidstate inducing accumulation or increase of the abnormal water content,was observed in the mass of the brain intracellularly or extracellularlyor both occasionally. The same model was used, with the cerebral edemaas the index, to study the effect of the chromanol glycoside inrepressing the morbid alteration of the hindrance of cerebral ischemicreperfusion.

[0086] Wister type male rats (body weights 250-300 g) were used for theexperiment as sorted into groups of 6 heads. The anesthesia, when theocclusion of the vertebral artery was elected, was effected byintraperitoneal administration injecting chloral hydrate(360 mg/kg).During the experiment, the body temperature of each rat was maintainedat 37° C. as rectum temperature by means of a warm mat and a warm rap.

[0087] Each of the rats was laid in the prone position, fixed by thehead on a rat fixing device, and cut open in a length of about 2 cmalong the median line on the posterior region of the neck. At thispoint, the rectus capitis posterior on the opposite sides were peeled atthe position of the second cervical vertebra with the aid of amicroscope (made by Nagashima Medical Instrument Co., Ltd., Japan) andthe opposite vertebral arteries were sought out in the occiput recti,peeled respectively from the connecting tissue, and exposed. With usinga microsurgery grade bipolar coagulator, the opposite vertebral arterieswere selectively electrocoagulated at the position of the secondcervical vertebra. Further, the vertebral arteries could be infalliblyoccluded by cutting the coagulated part with microsurgery grade scissorsand electrocoagulating the section. The treatment of the vertebralarteries was completed by suturing the ruptured skin with a silk thread.Then, the rat was set on the dorsal position and cut open longitudinallyin a length of about 2 cm along the median line in the anterior regionof the neck. Thereafter, the common carotid arteries on the oppositesides were exposed and peeled under the same microscope and they werestowed in the cut part by dint of a silk thread hooked thereon and thecut part was closed with a surgical staple. On the day following thesurgical experiment, the surgical staple was removed from the cutanterior part of the neck, the silk thread stowed in the cut part wasslightly pulled up, and the opposite common carotid arteries were closedlaterally toward each other by the use of a Sugita's clip (No. 52) forcerebral aneurism to form a four blood vessel occlusion model.

[0088] The prosencephalon ischemia was imparted to the rat by continuingthe occlusion of the opposite common carotid arteries for five minutesand, immediately before the start of the ischemic reperfusion, the TMGpreparation obtained in advance by thoroughly dissolving TMG inphysiological common salt solution at a concentration of 1.5 mg/ml wasadministered thereto by intravenous injection at a dose of 4 mg/kg ofbody weight. The reperfusion was started by removing the clip from thecommon carotid arteries. After the reperfusion had lasted for 120minutes, the rat was is immediately decapitated and the oppositecerebral hemispheres were promptly collected and weighed wet. Then, thecerebral hemispheres were dried in a drier at 100° C. for 48 hours andweighed dry. The difference of the weight before drying was found as thewater content. Then, the ratio of the water content relative to the wetweight of the brain (ratio of the brain water content) was calculated.

[0089] The results consequently obtained are shown in Table 6 inconjunction with the results obtained of the control group in which thephysiological common salt solution was administered in the same dose byintravenous injection immediately before the start of the ischemicreperfusion in the place of the TMG preparation. TABLE 6 Ratio of watercontent to brain (%) Control group 78.66 ± 0.51  TMG administrationgroup 78.05 ± 0.28* (4 mg/kg of body weight)

[0090] It is clearly noted from Table 6 that the TMG administrationgroup showed significant repression of the ratio of water content tobrain.

[0091] Test for Acute Toxicity

[0092] The agent of this invention for preventing and curing thecerebral edema due to the hindrances in the blood vessel was tested foracute toxicity with the view to confirming the safety of the use of theagent. The ICR type mice 4 to 5 weeks old were used in the test assorted into groups of three heads. As the chromanol glycoside, the sameTMG as mentioned above was suspended in an aqueous 5% gum arabicsolution and the suspension wad orally administered to the rats at arate of 500 mg/kg as reduced to TMG. The rats thus treated were placedunder observation for one week. In this case, to the rats of the controlgroup, an aqueous 5% gum arabic solution was administered orally at arate of 0.3 ml. None of the mice of the groups to which the preparationswere administered experienced death.

EXAMPLES OF PREPARATION Example 1 of Preparation

[0093] A powder was obtained by mixing 100 g of TMG, 800 g of milksugar, and 100 g of corn startch with a blender.

Example 2 of Preparation

[0094] A granular agent was obtained by mixing 100 g of TMG, 450 g ofmilk sugar, and 100 g of hydroxypropyl cellulose of a low degree ofsubstitution, kneading the resultant mixture with 350 g of an aqueous10% hydroxypropyl cellulose solution, granulating the produced blendwith an extrusion pelletizer, and drying the granules consequentlyformed.

Example 3 of Preparation

[0095] Tablets were obtained by mixing 100 g of TMG, 550 g of milksugar,215 g of corn starch, 130 g of crystalline cellulose, and 5 g ofmagnesium stearate in a blender, and molding the produced blend with atableting machine.

Example 4 of Preparation

[0096] A capsuled agent was obtained by mixing 10 g of TMG, 110 g ofmilk sugar, 58 g of corn starch, and 2 g of magnesium stearate in aV-shaped mixing machine and filling the capsules, No. 3, each with 180mg of the resultant-mixture.

Example 5 of Preparation

[0097] An injection agent was obtained by dissolving 200 mg of TMG and100 mg of glucose in 1 ml of purified water, filtering the solution,dispensing the filtrate among 2-ml ampoules, sealing the ampoules, andsterilizing the filled ampoules.

[0098] Industrial Utilizability

[0099] Since the agent of this invention for preventing and curing thehindrance of ischemic reperfusion has a chromanol glycoside as an activecomponent as described above, it is capable of reinforcing the in vivosystem for resisting oxidation, allowing effective repression andcontrol of active oxygen and free radicals in the part affected by thehindrance of ischemic reperfusion, conspicuously repressing a morbidalteration in the hindrance of ischemic reperfusion, and producing amarked improvement in eliminating the morbid state.

[0100] Further, since the agent of this invention uses as an activecomponent a chromanol glycoside possessing high water solubility, it canbe used as solid preparations and formulated as water preparationscontaining the active component at high concentrations. Thesepreparations, even at a small dosage, function effectively on theaffected part and prevent and cure the hindrance of ischemic reperfusionand, at the same time, warrant very safe use because it entails no sideeffect.

[0101] The agent of this invention for preventing and curing thehindrance of ischemic reperfusion can be utilized for preventing andcuring the hindrances induced in various parts such as heart, stomach,small intestine, liver, spleen, kidney, brain, eyeball, and skin by theischemic reperfusion and the hindrances incurred during thetransplantation of internal organs.

1. An agent for preventing and curing the hindrance of ischemicreperfusiton, having as an active component thereof a chromanolglycoside represented by the following general formula:

[wherein R¹, R², R³, and R⁴ independently denote a hydrogen atom or alower alkyl group, R⁵ denotes a hydrogen atom, a lower alkyl group, or alower acyl group, X denotes a monosaccharide residue or anoligosaccharide residue which may have a lower alkyl group or a loweracyl group substituted for the hydrogen atom of the hydroxyl group ofsaid saccharide residue, n denotes an integer of 0-6, and m denotes aninteger of 1-6].
 2. An agent according to claim 1, wherein saidchromanol glycoside is 2-(α-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol.
 3. An agent according to claim 1 or claim 2, wherein saidhindrance of ischemic reperfusion is a hindrance of small intestinalmucous membrane, a hindrance of cardiac muscle ischemic reperfusion, ora hindrance of the cerebral ischemic reperfusion.
 4. An agent accordingto any of claims 1-3, wherein said agent is an aqueous pharmaceuticalpreparation.